All viruses depend on host cells for their life cycle; thus, identifying the host factors required by each virus can reveal novel targets for antiviral development. We recently reported a novel quantitative proteomics-based strategy for comprehensively interrogating with high confidence molecular viral-host interactions and validated this pipeline with dengue virus (DV). DV is an important mosquito-borne virus that poses significant economic and public health burdens on much of the world and is also a potential threat to the US due to the recent geographical expansion of its mosquito vectors. The DV genome encodes only 10 proteins; thus, DV relies on these few proteins to recruit numerous host factors to replicate and complete its cycle of infection and reproduction. We focused on two DV nonstructural proteins that are essential for replication, NS5 and NS3. Our strategy identified ~850 unique high confidence candidates that interacted with NS5, NS3, or both. These proteins are thus promising candidates for host factors involved in DV replication. The challenge is to extend our previous discovery of these interacting proteins to efficiently determine which host proteins are critical for DV infection and how they perform their functions. We will comprehensively functionally validate our interaction datasets using a pooled shRNA gene knockdown approach. This work will yield a list of host factors, previously identified through thei interactions with NS5/NS3, which are important for DV infection. We will next test the highly innovative concept that interactions between viral proteins and host proteins reduce the functions of host proteins, generating viral-induced hypomorphs (vi-hypomorphs). The synthetic lethal/synthetic sick (SL) interaction partners of these vi-hypomorphs are thus also potential therapeutic targets, the inhibition of which is predicted to selectively kill infected cels. Using a comparative genomics strategy, we will predict and test the conservation of SL interactions from yeast to humans. As proof-of-principle, we will test whether the presence of the viral bait reduces the function of its host partners by targeting the SL partners of these putative vi- hypomorphs and determining whether cells expressing the viral bait are selectively killed. The exploratory approach we propose here will allow us to launch into detailed mechanistic studies of the molecular mechanisms by which DV establishes an environment favorable for infection, making this application highly suitable for the R21 funding mechanism. Moreover, our novel SL-based approach will potentially have a broad impact on the development of novel therapeutics against a wide range of intracellular pathogens.